CN110278946A - Thuringiensis microcapsules and preparation method thereof with uvioresistant ability - Google Patents
Thuringiensis microcapsules and preparation method thereof with uvioresistant ability Download PDFInfo
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- CN110278946A CN110278946A CN201910586384.2A CN201910586384A CN110278946A CN 110278946 A CN110278946 A CN 110278946A CN 201910586384 A CN201910586384 A CN 201910586384A CN 110278946 A CN110278946 A CN 110278946A
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- microcapsules
- nano
- thuringiensis
- ultraviolet
- wall material
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- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 239000002086 nanomaterial Substances 0.000 claims abstract description 109
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- 206010011732 Cyst Diseases 0.000 claims abstract description 34
- 208000031513 cyst Diseases 0.000 claims abstract description 34
- 229920000867 polyelectrolyte Polymers 0.000 claims abstract description 23
- 101710151559 Crystal protein Proteins 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims description 148
- 238000000034 method Methods 0.000 claims description 41
- 239000000725 suspension Substances 0.000 claims description 41
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 38
- 235000010413 sodium alginate Nutrition 0.000 claims description 31
- 239000011162 core material Substances 0.000 claims description 29
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 102000004169 proteins and genes Human genes 0.000 claims description 29
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 28
- 239000000661 sodium alginate Substances 0.000 claims description 28
- 229940005550 sodium alginate Drugs 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
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- 150000001875 compounds Chemical class 0.000 claims description 13
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 12
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 12
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- 239000007788 liquid Substances 0.000 claims description 12
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- 238000009938 salting Methods 0.000 claims description 10
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- 229940010747 sodium hyaluronate Drugs 0.000 claims description 9
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 9
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 7
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- 239000006097 ultraviolet radiation absorber Substances 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
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- 229940083542 sodium Drugs 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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- 231100000765 toxin Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
- A01N25/28—Microcapsules or nanocapsules
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a kind of thuringiensis microcapsules and preparation method thereof with uvioresistant ability.The microcapsules be with thuringiensis crystal proteins (such as, Cry1Ac crystal) it is core, it selects the polyelectrolyte of two kinds of oppositely chargeds under preparation condition to form cyst wall on thuringiensis crystal proteins surface by LBL self-assembly, and is made in cyst wall layer outer surface load anti-ultraviolet nano material layer.The microcapsules have stronger insecticidal power, longer-term effect and preferable resistant to high temperatures, resist drying and uvioresistant stability.
Description
Technical field
The invention belongs to agricultural biological preparation technique fields, are related to a kind of Su Yunjin brood cell's bar with uvioresistant ability
Bacteria microcapsule and preparation method thereof.
Background technique
Thuringiensis (Bacillus thuringiensis, Bt) is a kind of generally existing Gram-positive
Aerobic bacteria belongs to one in " primary Jie Shi Bacteria Identification handbook " the 9th edition in the 18th group of Bacillus of the second class
Kind.Different from other nearly edge bacillus is a bit, Bt while generating brood cell, can also generate diamond shape, biconial,
The insecticidal crystal protein of the shapes such as elliposoidal, rectangle and irregular shape, i.e. parasporal crystal.
Parasporal crystal is by one or more Cytolitic (Cyt) toxin protein and Crystal (Cry) toxin protein group
At.For every kind of parasporal crystal all only specifically for a kind of narrow spectrum insect, main insecticidal spectrum has Lepidoptera, Homoptera, straight wing
The various insects such as mesh, Hymenoptera, Isoptera, coleoptera and nematode and protozoan etc..This species specificity is by Cry toxin
It is determined with the specific binding for being located at insect midgut cell surface receptor protein.Meanwhile Bt is when playing insecticidal action, it is right
Human body, vertebrate and non-phytotoxic belong to environmentally friendly biological insecticides.Therefore, Bt has existed as a kind of biological pesticide
Agricultural, forestry and mosquito-proof field apply more than 80 years, are current biological pesticides most widely used in the world.But after its use
Shorter lasting effect constrains the commercial applications of Bt, and the environmental factors such as ultraviolet radioactive, drying, high temperature can all lead to its activity
The inactivation of ingredient insecticidal crystal protein.In order to improve the lasting effect of Bt, different bacillus thuringiensis is had developed both at home and abroad
Dosage form mainly has oil-suspending agent, microcapsules, floating block agent, oil aerosol, ultraviolet protection dosage form, aqueous suspension agent and soil
Mineral grain adsorbent type etc..
In recent years, Micro-Encapsulation Technique is grown rapidly.Microcapsules be by certain wall-forming materials by the intracapsular external space every
From poor environment interference to protect intracapsular active constituent, is protected it from, reaches and extend lasting effect, enhancing stability, targeted delivery
The purpose of with immunological rejection is prevented.It is to improve its resistance to environment-stress that microcapsule formulations, which are made, in bioactive ingredients
Common method.The self-assembling technique of microcapsules be based on polyelectrolyte zwitterion with interacting between positive and negative charge
A kind of technology.The substrate surface for being mainly characterized by charging on surface of the technology is alternately adsorbed by electrostatic interaction
The polyelectrolyte of oppositely charged.The micro-capsule that this method obtains has various excellent compared with traditional micro-capsule
More property: its preparation process is simple；Preparation condition is mild, can carry out in room temperature aqueous solution, it is ensured that biomolecule maintains life
Active native conformation of object etc..
In the previous work in laboratory, we are with positively charged chitosan (Chitosan, Cs) and negatively charged
Sodium alginate (sodium alginate, Alg) is wall material, by layer-by-layer, is prepared by core of insecticidal proteins
A kind of microcapsules.The microcapsules have ability resistant to high temperatures and dry, but the performance in uvioresistant is not satisfactory.
Therefore, presently, there are the problem of be to need to research and develop a kind of to there is stronger insecticidal power, longer-term effect and preferably
The thuringiensis insecticide of resistant to high temperatures, resist drying and uvioresistant stability.
Summary of the invention
It is a kind of with uvioresistant ability the technical problem to be solved by the present invention is to provide in view of the deficiencies of the prior art
Thuringiensis microcapsules and preparation method thereof.The microcapsules are with thuringiensis crystal proteins (example
Such as, Cry1Ac crystal) it is core, select the polyelectrolyte of two kinds of oppositely chargeds under preparation condition to pass through LBL self-assembly
Cyst wall is formed, and is made in cyst wall layer surface load anti-ultraviolet nano material layer.The microcapsules have stronger insecticidal power,
Longer-term effect and preferably resistant to high temperatures, resist drying and uvioresistant stability.
For this purpose, first aspect present invention provides a kind of thuringiensis microcapsules with uvioresistant ability,
By the thuringiensis crystal proteins as core, the cyst wall that is coated on outside core, and it is adsorbed in cyst wall outer surface
Anti-ultraviolet nano material layer constitute.
According to the present invention, mass ratio >=0.3:1 of the anti-ultraviolet nano material and thuringiensis microcapsules,
Preferably 0.3:1.
In the present invention, the anti-ultraviolet nano material includes nano-TiO2, nano-ZnO and Nano-meter SiO_22One of or it is several
Kind.
In some embodiments of the invention, the thuringiensis microcapsules uvioresistant time >=2h, preferably >=
8h。
In some embodiments of the invention, the thuringiensis crystal proteins are Cry1Ac crystal.
In the present invention, the cyst wall is by the polyelectrolyte macromolecular with positive charge and with the polyelectrolyte of negative electrical charge
Macromolecular is formed as the layer-by-layer alternating sorbent of wall material in the outer surface of core；Wherein, the polyelectrolyte with positive charge is big
Molecule includes chitosan and/or poly-D-lysine with positive charge, the preferably chitosan of positive charge；It is described to have negative electricity
The macromolecular of lotus includes one in negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate
Kind or several, preferably negatively charged sodium alginates.
In some embodiments of the invention, the wall material number of plies N of the cyst wall is the integer more than or equal to 3, preferably institute
The wall material number of plies N for stating cyst wall is the integer of 3-12, and the wall material number of plies N of the further preferred cyst wall is the integer of 3-10.
In the present invention, the partial size of the microcapsules is (0.25-0.50 μm) × (0.60-1.00 μm).
In some embodiments of the invention, the anti-ultraviolet nano material is nano-ZnO and/or nano-TiO2, N
Layer wall material include one of negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate or
Several, preferably negatively charged sodium alginates.
In other embodiments of the invention, the anti-ultraviolet nano material is Nano-meter SiO_22, n-th layer wall material includes
Chitosan and/or poly-D-lysine with positive charge, the preferably chitosan of positive charge.
Second aspect of the present invention provides the thuringiensis microcapsules of one kind as described in the first aspect of the invention
Preparation method comprising the step of loading anti-ultraviolet nano material: the wall material number of plies is micro- for the thuringiensis of N-1 or N
Capsule is mixed with the suspension containing anti-ultraviolet nano material, stirring, obtained to load the Su Yunjin bud for having anti-ultraviolet nano material
Born of the same parents' bacillus microcapsules.
In some embodiments of the invention, anti-ultraviolet nano material in the suspension containing anti-ultraviolet nano material
The content of material is 10g/L.
In some embodiments of the invention, the matter of the anti-ultraviolet nano material and thuringiensis microcapsules
Measure ratio >=0.3:1, preferably 0.3:1.
According to certain embodiments of the present invention, the wall material number of plies is N, and the anti-ultraviolet nano material is nano-ZnO
And/or Nano-meter SiO_22, the suspension containing anti-ultraviolet nano material is dispersed in water by anti-ultraviolet nano material to be formed.
In some embodiments of the invention, the anti-ultraviolet nano material is nano-ZnO, and n-th layer wall material includes that band is negative
One or more of sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate of charge, preferably band
The sodium alginate of negative electrical charge, the pH value of the suspension containing anti-ultraviolet nano material are the time of the stirring in 7.0
For 2h.
In other embodiments of the invention, the anti-ultraviolet nano material is Nano-meter SiO_22, n-th layer wall material includes
Chitosan and/or poly-D-lysine with positive charge, the preferably chitosan of positive charge, it is described to contain anti-ultraviolet nano material
The pH value of the suspension of material is in 4.0, and the time of the stirring is 2h.
Other embodiments according to the present invention, the wall material number of plies are N-1, and the anti-ultraviolet nano material is to receive
Rice TiO2, the suspension containing anti-ultraviolet nano material is by anti-ultraviolet nano material and the dispersion of the compound of n-th layer wall material
Yu Shuizhong is formed.
In some embodiments of the invention, the n-th layer wall material is to include with the chitosan of positive charge and/or more
Polylysine, the preferably chitosan of positive charge, N-1 layers of wall material include negatively charged sodium alginate, carboxymethyl cellulose
One or more of sodium, chondroitin sulfate and Sodium Hyaluronate, preferably negatively charged sodium alginate are described containing anti-
The pH value of the suspension of ultraviolet nanometer material is 5.0, and the time of the stirring is 2h.
In some embodiments of the invention, in anti-ultraviolet nano material and the compound of n-th layer wall material, the anti-purple
The mass ratio of outer nano material and n-th layer wall material is 1:1.
According to the present invention, the method also includes the wall material number of plies is prepared before the step of loading anti-ultraviolet nano material
For N-1 or N thuringiensis microcapsules the step of comprising:
Step A, will be as the polyelectrolyte macromolecular for having positive charge or negative electrical charge of wall material and as the first core material
Thuringiensis crystal proteins are uniformly mixed in salting liquid, concussion, outside thuringiensis crystal proteins
Surface forms first layer wall material, after further separating treatment, washing, obtains the micro- glue of thuringiensis that the wall material number of plies is 1
Capsule；
Step B with the polyelectrolyte macromolecular with the wall material opposite charges in previous step and will make as wall material
The thuringiensis microcapsules that the wall material number of plies for the second core material is 1 are uniformly mixed in salting liquid, concussion, in wall material
The thuringiensis microcapsule exterior surface that the number of plies is 1 forms second layer wall material, after further separating treatment, washing, obtains
The thuringiensis microcapsules that the wall material number of plies is 2；
Step C is repeated in step B N-2 times, is often repeated once then one layer of wall material of increase, is N thus to obtain the wall material number of plies
Thuringiensis microcapsules；
Wherein, the pH value of the salting liquid is 4.5-6.0, preferably 4.5-5.
Third aspect present invention provides thuringiensis microcapsules or such as sheet as described in the first aspect of the invention
Application of the thuringiensis microcapsules of the preparation of method described in invention second aspect in control of agricultural pest.
The present inventor selects the polyelectrolyte of two kinds of oppositely chargeds under preparation condition big using layer-by-layer
Successively alternating sorbent obtains in the parasporal crystal surface of Bt formation cyst wall and contains Bt molecule (such as chitosan and sodium alginate)
Parasporal crystal and with good controlled release properties microcapsules.The present inventor further selects nano-TiO2, nano-ZnO, nanometer
SiO2These three nano particles, the feature nontoxic using its, tasteless, pollution-free, absorpting and scattering ultraviolet capability is strong will
Its outer surface that microcapsules outermost layer cyst wall is adsorbed on by interaction between positive and negative charge, is made with uvioresistant ability
Thuringiensis microcapsules.The thuringiensis microcapsules with uvioresistant ability, which remove, has stronger desinsection effect
Power, longer-term are imitated, outside preferable resistance, also have preferable resistant to high temperatures, resist drying and uvioresistant stability.
Detailed description of the invention
It is next with reference to the accompanying drawing that invention is further described in detail:
Fig. 1 shows the form of microcapsules under scanning electron microscope；Wherein, A is Cry1Ac crystal (× 50000)；B is
Cry1Ac crystal microcapsules (× 50000).
Fig. 2 shows the microencapsulated forms (× 50000) when loading different nano particles under scanning electron microscope；Wherein,
A is ZnO and (Cs(95%DD)/Alg)5Microcapsules pH 7.0；B is SiO2With (Alg/Cs(95%DD)) 5Microcapsules pH 4.0；C is
TiO2- Cs and (Cs(95%DD)/Alg)5Microcapsules.
Fig. 3 shows the 50mM through pH 10.2 after the thuringiensis microcapsules ultraviolet irradiation with uvioresistant ability
Na2CO3The SDS-PAGE characterization result for the supernatant being centrifuged after solution processing；Wherein, A is Cry1Ac crystal；B is load SiO2
Microcapsules；C is the microcapsules of loading ZnO；D is load TiO2Microcapsules；1 is Marker；2 is without ultraviolet irradiations；3 are
Ultraviolet irradiation 2h；4 be ultraviolet irradiation 4h；5 be ultraviolet irradiation 6h；6 be ultraviolet irradiation 8h.
Specific embodiment
To be readily appreciated that the present invention, below in conjunction with drawings and examples, the present invention will be described in detail.But it is retouching in detail
Before stating the present invention, it should be understood that the present invention is not limited to the specific embodiments of description.It is also understood that art used herein
Language is not offered as restrictive only for describing specific embodiment.
Unless otherwise defined, all terms used herein and those skilled in the art's is logical
Understand meaning having the same.Although similar or equivalent any method and material with method described herein and material
It can also be used in implementation or test of the invention, but preferred method and material will now be described.
I, term
Heretofore described term " larva ", which refers to, to be had much form and physilogical characteristics and has Embryo in insect ontogeny
The stage of development of matter.
Term " active constituent " of the present invention refers to the ingredient for having killing or inhibiting effect to insect or its larva.
Heretofore described term " thuringiensis crystal proteins " refers to killing from thuringiensis
Worm crystalline protein, i.e. parasporal crystal are primarily referred to as Crystal (Cry) toxin protein.
Heretofore described " water " word, deionized water, distilled water are referred in the case where being not particularly illustrated or limiting
Or ultrapure water.
Heretofore described term " the cyst wall number of plies " and " the wall material number of plies " may be used interchangeably.
II, embodiment
As mentioned earlier, as a kind of widely used biological pesticide, Bt plays exactly its insecticidal crystal egg of toxicity action
White or its protein aggregate (parasporal crystal), but it exists such as in the application that desinsection speed is slow, the residual period is short, vulnerable to
The problems such as such environmental effects such as ultraviolet radioactive, drying, high temperature.Moreover, in the previous work in laboratory, with positively charged
Chitosan (Cs) and negatively charged sodium alginate (sodium alginate, Alg) be wall material, pass through LBL self-assembly skill
Art, although there is ability resistant to high temperatures and dry using the microcapsules that insecticidal proteins are prepared as core, the table in uvioresistant
It is existing not satisfactory.In consideration of it, the present inventor imitates the preparation method, drug effect, phase of thuringiensis insecticide, especially
It is that anti-ultraviolet radiation stability etc. has carried out a large amount of research.
Good radiation-proof effect can be obtained the study found that ultraviolet absorber is directly mixed with Bt preparation in the present inventor.
In order to preferably realize ultraviolet absorber for the cladding of thuringiensis microcapsules, the present inventors have noted that nanometer material
Material.Nano material refers to size in the material of 1nm-100nm, when Nano grade size is made in common material, its physics,
Chemical property can occur to change accordingly, such as can generate quantum size effect, small-size effect, skin effect and maroscopic quantity
Sub- tunnel-effect etc..And so that nanoparticle is had uvioresistant ability is exactly small-size effect, i.e., when the size of nano particle is close
When like or less than the wavelength of light wave, the coherence length of superconducting state or transmission depth, the de Broglie wavelength of conduction electronics, boundary
The periodicity of condition is destroyed, and the superficial layer atomic density of amorphous nano particle reduces, and can cause Material Physics, chemically
The variation of matter leads to the change of sound, light, mechanics, electromagnetism, macroscopic property etc., and material is made to have the ability for absorbing ultraviolet light.
The present inventor passes through research selection nano-TiO2, nano-ZnO, Nano-meter SiO_22These three nano particles, nontoxic using its, tasteless,
It is adsorbed on microcapsules by interaction between positive and negative charge by feature pollution-free, absorpting and scattering ultraviolet capability is strong
The outer surface of cyst wall has been successfully made the thuringiensis microcapsules with efficient uvioresistant ability, and based on this completion
The present invention.
Therefore, there are the thuringiensis microcapsules of uvioresistant ability involved in first aspect present invention, by
Thuringiensis crystal proteins, the cyst wall that is coated on outside core as core, and it is adsorbed in cyst wall outer surface
Anti-ultraviolet nano material layer is constituted.
In the thuringiensis microcapsules with uvioresistant ability, the cyst wall is by the poly- electricity with positive charge
Solve matter macromolecular and polyelectrolyte macromolecular with negative electrical charge as the layer-by-layer alternating sorbent of wall material core outer surface shape
At, and the anti-ultraviolet nano material is to be adsorbed on the outer surface of effect microcapsule wall by interacting between positive and negative charge.Also
It is to say, has embedded thuringiensis crystal proteins in the cyst wall of each microcapsules, and the outermost layer capsule of each microcapsules
The outer surface load of wall has anti-ultraviolet nano material.It is to be understood that each with Su Yunjin brood cell's bar of uvioresistant ability
Bacteria microcapsule includes: the thuringiensis crystal proteins as active constituent, outside active constituent by with just and
The cyst wall that the polyelectrolyte macromolecular of negative electrical charge is successively alternatively formed as wall material, and by interacting between positive and negative charge
It is adsorbed on the anti-ultraviolet nano material layer of effect microcapsule wall outer surface.
Obviously, anti-ultraviolet nano material is the thuringiensis microcapsules uvioresistant that control has uvioresistant ability
The key factor of ability.In the present invention, the anti-ultraviolet nano material includes nano-TiO2, nano-ZnO and Nano-meter SiO_22In
One or more, preferably nano-ZnO.
In the present invention, mass ratio >=0.3:1 of the anti-ultraviolet nano material and thuringiensis microcapsules is excellent
It is selected as 0.3:1.It is to be understood that every gram of thuringiensis microcapsules (Su Yunjin of unsupported anti-ultraviolet nano material
Bacillus microcapsules) anti-ultraviolet nano material load amount >=300mg, preferably 300mg；Also that is, anti-ultraviolet nano material
In thuringiensis microcapsules (the thuringiensis microcapsules of unsupported anti-ultraviolet nano material), outer surface is most
Whole adsorption concentration is >=300mg/g, preferably 300mg/g.
By to having loaded TiO2, Nano-meter SiO_22Microcapsules with ZnO are irradiating after 8h, then sample is dissolved in alkalinity
Supernatant is taken to carry out SDS-PAGE characterization after being centrifuged in solution, the results showed that, these three nanoparticles all play crystalline protein
The protective action of ultraviolet light irradiation, wherein strongest ultraviolet protection effect is nano-ZnO, Nano-meter SiO_22Take second place, and nanometer
TiO2Effect is slightly poorer to the above two.And the thuringiensis microcapsules uvioresistant time provided by the present invention in 2h or more,
8h or more can be even easily reached.
In the present invention, wall material be limit microcapsules application another restraining factors, especially using microcapsule formulation as
In the case where insecticide, needs cheap and/or substance with biocompatibility as wall material, just can make microcapsules more
Has use value.Sodium alginate is a kind of linear anionic natural polysaecharides compound, mainly from bulk kelp, sargassum and
It is extracted in the sea-plants such as kelp.The pKa of sodium alginate is 3.38-3.65, has great amount of hydroxy group and carboxyl, shape on strand
Although at microcapsules permeability it is very low, pH easily influences its dissolubility.Currently, the most common mode is in sodium alginate table
Face deposits one layer of new cationic polymer wall material, such as chitosan.Chitosan is uniquely largely deposited known to current nature
Alkaline weak cation polysaccharide, pKa is about 6.3, can with negatively charged polyelectrolyte because institute it is electrically charged on the contrary,
To interact because of electrostatic force, polyelectrolyte multilayer composite micro-capsule is formed, can be played in terms of loading
Effect.
In the present invention, the polyelectrolyte macromolecular with positive charge includes with the chitosan of positive charge and/or more
Polylysine, the preferably chitosan of positive charge；The macromolecular with negative electrical charge include negatively charged sodium alginate,
One or more of sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate, preferably negatively charged alginic acid
Sodium.
It is known in the art that chitosan is that chitin (chitin) sloughs the day for preparing formation after 50% or more acetyl group
Right macromolecule polysaccharide.Degree of deacetylation is different, then primary amino group number is different in chitosan molecule chain.Deacetylation (DD) is
The important parameter of chitosan material can influence its physicochemical property (solubility, crystallinity, swelling behavior, mechanical performance) and life
Object characteristic (protein adsorption ability).Deacetylation is higher, and the tensile strength and elasticity of chitosan are higher, but its brittleness
It is higher.Chitosan employed in the present invention includes the chitosan of 90%DD and/or the chitosan of 95%DD.
If those skilled in the art will be appreciated that cyst wall, the number of plies is less, and Jacket thickness will be relatively thin, in this way to egg
The protection of white matter will reduce, and controlled-release effect is also bad.And if the cyst wall number of plies is excessive, it not only will affect drug effect, also can
Increase unnecessary workload, and will increase cost.And the active constituent under the suitable cyst wall number of plies, in capsule
Can either be valid protected, but can under the controlled-release function of cyst wall useful effect in target spot.Therefore, of the invention some
In embodiment, the wall material number of plies N of the cyst wall is the integer more than or equal to 3, and the wall material number of plies N of the preferably described cyst wall is 3-
The wall material number of plies N of 12 integer, the further preferred cyst wall is the integer of 3-10.
It is noted that the present inventor by numerous studies find, the anti-ultraviolet nano material be nano-ZnO and/
Or nano-TiO2, n-th layer wall material selects negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and hyalomitome
One or more of sour sodium, preferably negatively charged sodium alginate, UVResistant effect are best.And when the uvioresistant is received
Rice material is Nano-meter SiO_22, chitosan and/or poly-D-lysine of the selection of n-th layer wall material with positive charge, preferably positive charge
Chitosan, UVResistant effect is best.
In some specific preferred embodiments of the invention, the partial sizes of the microcapsules be (0.25-0.50 μm) ×
(0.60-1.00μm).The Microcapsules Size is essentially the partial size of thuringiensis crystal proteins, that is to say, that Su Yun
The wall material of golden bacillus microcapsules is little to the grain diameter influence of microcapsules.With the microcapsules phase of many template etching methods preparation
Than Microcapsules Size of the invention is smaller, is more suitable as insecticide to spray for insect larvae feeding and field.
The system of thuringiensis microcapsules involved in second aspect of the present invention as described in the first aspect of the invention
Preparation Method mainly includes the steps that loading anti-ultraviolet nano material: the wall material number of plies is micro- for the thuringiensis of N-1 or N
Capsule is mixed with the suspension containing anti-ultraviolet nano material, stirring, obtained to load the Su Yunjin bud for having anti-ultraviolet nano material
Born of the same parents' bacillus microcapsules.
For the present inventor the study found that other than the property of anti-ultraviolet material itself, the loading condition of anti-ultraviolet material is shadow
Ring the another factor of the uvioresistant performance of thuringiensis microcapsules.For example, the present invention is further ground by a large number of experiments
Study carefully discovery, the optimum load condition of nano-ZnO is neutral environment (for example, pH value is 7.0) and microcapsules outermost material is
Alg；Nano-meter SiO_22Optimum load condition be acidic environment (for example, pH value is 4.0) and microcapsules outermost material is Cs；
And nano-TiO2Optimum load condition be acidic environment (for example, pH value be 5.0), TiO2It forms compound with Cs and improves and disperse
Property and microcapsules outermost material be Alg.
Specifically, the loading condition of anti-ultraviolet material of the invention is as follows:
(1) when the anti-ultraviolet nano material is nano-ZnO, the micro- glue of thuringiensis for being N by the wall material number of plies
Capsule is mixed with the suspension containing anti-ultraviolet nano material, stirring, obtained to load the Su Yunjin brood cell for having anti-ultraviolet nano material
Bacillus microcapsules.
N-th layer wall material includes negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and hyaluronic acid
One or more of sodium, preferably negatively charged sodium alginate (Alg), the suspension containing anti-ultraviolet nano material
The pH value of liquid is 7.0, and the time of the stirring is 2h.
The suspension containing anti-ultraviolet nano material is dispersed in water shape by anti-ultraviolet nano material (nano-ZnO)
At the content of anti-ultraviolet nano material (nano-ZnO) is 10g/L in the suspension containing anti-ultraviolet nano material；It is described
Mass ratio >=the 0.3:1 for the thuringiensis microcapsules that anti-ultraviolet nano material (nano-ZnO) and the wall material number of plies are N, it is excellent
It is selected as 0.3:1；Also that is, the anti-ultraviolet nano material (nano-ZnO) is in the final of thuringiensis microcapsule exterior surface
Adsorption concentration >=300mg/g, preferably 300mg/g.
(2) when the anti-ultraviolet nano material is Nano-meter SiO_22When, the wall material number of plies is micro- for the thuringiensis of N
Capsule is mixed with the suspension containing anti-ultraviolet nano material, stirring, obtained to load the Su Yunjin bud for having anti-ultraviolet nano material
Born of the same parents' bacillus microcapsules.
N-th layer wall material includes chitosan and/or poly-D-lysine with positive charge, and the preferably shell of positive charge is poly-
(Cs), the pH value of the suspension containing anti-ultraviolet nano material is 4.0, and the time of the stirring is 2h.
The suspension containing anti-ultraviolet nano material is by anti-ultraviolet nano material (Nano-meter SiO_22) it is dispersed in water shape
At anti-ultraviolet nano material (Nano-meter SiO_2 in the suspension containing anti-ultraviolet nano material2) content be 10g/L；Institute
State anti-ultraviolet nano material (Nano-meter SiO_22) with the wall material number of plies be N thuringiensis microcapsules mass ratio >=0.3:
1, preferably 0.3:1；Also that is, the anti-ultraviolet nano material (Nano-meter SiO_22) in thuringiensis microcapsule exterior surface
Final adsorption concentration >=300mg/g, preferably 300mg/g.
(3) when the anti-ultraviolet nano material is nano-TiO2When, the thuringiensis for being N-1 by the wall material number of plies
Microcapsules are mixed with the suspension containing anti-ultraviolet nano material, stirring, obtained to load the Su Yunjin for having anti-ultraviolet nano material
Bacillus microcapsules.
N-1 layers of wall material include negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and hyalomitome
One or more of sour sodium, preferably negatively charged sodium alginate (Alg)；The n-th layer wall material is to include with positive electricity
The chitosan and/or poly-D-lysine of lotus, the preferably chitosan (Cs) of positive charge, it is described containing anti-ultraviolet nano material
The pH value of suspension is 5.0, and the time of the stirring is 2h.Thus thuringiensis microcapsules outermost layer obtained
Cyst wall is chitosan, and load has nano-TiO in outermost shell glycan cyst wall outer surface2Layer.
The suspension containing anti-ultraviolet nano material is by anti-ultraviolet nano material (nano-TiO2) and n-th layer wall material
The compound of (such as Cs) is dispersed in water to be formed, anti-ultraviolet nano material in the suspension containing anti-ultraviolet nano material
Expect (nano-TiO2) content be 10g/L；Anti-ultraviolet nano material (the nano-TiO2) and thuringiensis microcapsules
Mass ratio >=0.3:1, preferably 0.3:1；Also that is, the anti-ultraviolet nano material (nano-TiO2) in Su Yunjin brood cell's bar
Final adsorption concentration >=the 300mg/g, preferably 300mg/g of bacteria microcapsule outer surface.
Those skilled in the art will be appreciated that when the anti-ultraviolet nano material is nano-TiO2When, due to nanometer
TiO2The dispersibility of particle is bad, and agglomeration easily occurs in adsorption process and cannot be adsorbed onto surface of microcapsule.So making
Use nano-TiO2When as anti-ultraviolet nano material, using with above two anti-ultraviolet material (nano-TiO and Nano-meter SiO_22) no
With method, n-th layer wall material (Cs) used is as nano-TiO using in the present invention2The dispersing agent of particle, makes itself and nano-TiO2Shape
At compound, and it is further formed the suspension containing anti-ultraviolet nano material.It is thus obtained to contain anti-ultraviolet nano material
Suspension in actually containing n-th layer wall material (Cs) be nano-TiO2Compound (the nano-TiO of particle2-Cs)。
In some specific examples, the preparation method of the suspension containing anti-ultraviolet nano material includes: by TiO2
It is dissolved in deionized water with n-th layer wall material (such as Cs), under room temperature magnetic agitation 6h, adjusts pH value to 5.0, be made and contain
There is the suspension of anti-ultraviolet nano material；Be readily appreciated that, in the suspension actually containing n-th layer wall material (Cs) be nanometer
TiO2Compound (the nano-TiO of particle2- Cs), and in anti-ultraviolet nano material (nano-TiO2) and n-th layer wall material (for example,
Cs in compound), the anti-ultraviolet nano material (nano-TiO2) it with the mass ratio of n-th layer wall material (for example, Cs) is 1:1.
In some further specific examples, for example, by 2.7g TiO2, 2.7g Cs is dissolved in 1L deionized water, room
Magnetic agitation 6h under the conditions of temperature adjusts pH value to 5.0, makes containing TiO2With the suspension for the compound that Cs mass ratio is 1:1
Liquid.
According to the present invention, the method also includes the wall material number of plies is prepared before the step of loading anti-ultraviolet nano material
For N-1 or N thuringiensis microcapsules the step of comprising:
Step A, by the polyelectrolyte macromolecular (for example, chitosan) or negative electrical charge as wall material with positive charge
Polyelectrolyte macromolecular (for example, sodium alginate) is with the thuringiensis crystal proteins as the first core material in pH value
For 4.5-6.0, preferable ph 4.5-5, further preferred pH value is is uniformly mixed in 5 salting liquid, concussion, in Su Yunjin
Bacillus crystal proteins outer surface forms first layer wall material, and after further separating treatment, washing, obtaining the wall material number of plies is 1
Thuringiensis microcapsules；
Step B, by the polyelectrolyte macromolecular (example having with the wall material opposite charges in previous step as wall material
Such as, sodium alginate or chitosan) with as the second core material the wall material number of plies be 1 thuringiensis microcapsules precipitating
Object is 4.5-6.0 in pH value, and preferable ph 4.5-5, further preferred pH value is is uniformly mixed in 5 salting liquid, concussion,
Second layer wall material, further separating treatment, washing are formed in the thuringiensis microcapsule exterior surface that the wall material number of plies is 1
Afterwards, the thuringiensis microcapsules that the wall material number of plies is 2 are obtained；
Step C is repeated in step B N-2 times, is often repeated once then one layer of wall material of increase, is N thus to obtain the wall material number of plies
Thuringiensis microcapsules.
In some specific preferred embodiments of the invention, the salting liquid is the sodium chloride that molar concentration is 0.5M
Aqueous solution.
In some specific preferred embodiments of the invention, concentration of the wall material in salting liquid is 0.1 g/L.
In some embodiments of the invention, mass ratio >=1:1 that wall material is mixed with core material, it is preferable that wall material and core material
Mixed mass ratio is 1:(1-2).
According to the method for the present invention, in step A and step B, time >=20 minute of the concussion, preferably 20 minutes.
It is not special for the method for separating and processing of the thuringiensis microcapsules in step A-C in the present invention
Limitation can use the separation method of this field routine.For example, in step A-C, using centrifugal separation to Su Yunjin bud
Born of the same parents' bacillus microcapsules carry out separating treatment, and the revolving speed of the centrifugation is 1500-6000rpm, preferably 2500-6000rpm, into
One step is preferably 6000rpm, and the time of the centrifugation is 10-60 minutes, preferably 10-30 minutes, further preferably 10 points
Clock.
In certain embodiments, contain the Soviet Union in thuringiensis microcapsules mixed liquor in step A-C
Cloud gold bacillus microcapsules successively carry out separating treatment and washing at least 2 times.
Third aspect present invention provides thuringiensis microcapsules or such as sheet as described in the first aspect of the invention
Application of the thuringiensis microcapsules of the preparation of method described in invention second aspect in control of agricultural pest.Institute
Stating agricultural pest includes the insect that all thuringiensis effectively can inhibit or kill, especially these insects
Larva.
In some embodiments of the invention, the worm includes isoptera insect, Homoptera insect, orthopteran, film
The larva of one or more of homopterous insect, lepidopterous insects, coleopteron, nematode and protozoan, preferably elytrum
The larva of mesh insect, further preferably anomala corpulenta larva.
III, embodiment
The present invention is specifically described below by way of specific embodiment.Experimental method described below, such as without special theory
It is bright, it is laboratory conventional method.Experimental material described below can be obtained unless otherwise instructed by commercial channel.
In the present invention size and form of microcapsules using scanning electron microscope observe: take Bt microcapsules sample, go from
Sub- water, which is diluted to after debita spissitudo, respectively takes 20 μ L to be added dropwise on clean aluminium-foil paper, and sputter coating machine pair is utilized after natural drying
Sample metal spraying is observed micro- using scanning electron microscope (JSM-6700F cold field emission scanning electron microscope, Japan Electronics)
The size and form of capsule.
SDS-PAGE is carried out to sample using BIO-RAD electrophoresis apparatus (Bio Rad Laboratories) in the present invention and carries out SDS-
PAGE characterization.
Embodiment 1: using Cry1Ac crystal as core preparation have uvioresistant ability thuringiensis microcapsules 1,
Prepare microcapsules reagent preparation
0.5M NaCl solution: 58.5g NaCl is added deionized water and is settled to 2L；
100mM Tris buffer: 121g Tris is added deionized water and is settled to 1L；
The Cs of the Cs:1g 95%DD of 1.0mg/mL 95%DD is dissolved in 1L 0.5M NaCl solution, magnetic agitation 6h,
PH value is adjusted to 5.0；
1.0mg/mL Alg:1g Alg is dissolved in 1L 0.5M NaCl solution, magnetic agitation 18h, adjusts pH value to 5.0；
Nano-TiO2With the compound (TiO of Cs2- Cs): 2.7g TiO2, 2.7g Cs is dissolved in 1L deionized water, room temperature
Under the conditions of magnetic agitation 6h, adjust pH value to 5.0；
Nano-ZnO suspension: it takes ZnO Nanoparticles (50 ± 10nm) to be dissolved in the water that pH is 4.0,7.0, is stirred in magnetic force
It mixes and stirs 30min on device, be prepared into the suspension of 10g/L；
Nano-meter SiO_22Suspension: Nano-meter SiO_2 is taken2Particle (30 ± 10nm) is dissolved in the water that pH is 4.0,7.0, is stirred in magnetic force
It mixes and stirs 30min on device, be prepared into the suspension of 10g/L.
2, preparation has the thuringiensis microcapsules of uvioresistant ability
(1) the Cry1Ac crystal microcapsules (thuringiensis that the LBL self-assembly method preparation wall material number of plies is N-1 or N
Microcapsules)
The crude extract of 50mL Cry1Ac crystal brood cell's mixture is taken, is centrifuged, 6000rpm 10min abandons supernatant.
First layer: it takes 50mL 1.0mg/mL Cs (95%DD) and structure cell mixture precipitation to mix, vibrates 20 min.From
The heart, 6000rpm, 10min abandon supernatant.It will be centrifuged after precipitating washing, 6000rpm, 10min abandon supernatant, be repeated 3 times；
The second layer: it takes 50mL 1.0mg/mL Alg and above-mentioned precipitating to mix, vibrates 20min.Centrifugation, 6000 rpm,
10min abandons supernatant.It will be centrifuged after precipitating washing, 6000rpm, 10min abandon supernatant, repeat 3 times；
The packaging method of 3-10 layers of wall material is identical as the 1st, 2 layer of packaging method, the sequence of wall material from inside to outside are as follows: Cs-
Alg-Cs-Alg-Cs-Alg-Cs-Alg-Cs-Alg.The method is available to have wrapped up 10 layers of wall by core of Cry1Ac crystal
(Cs (95%DD)/Alg) of material5Microcapsules, by its structure of scanning electron microscopic observation, as shown in Figure 1B.
The material of first floor wall material and second layer wall material is exchanged, it is available with same method to be with Cry1Ac crystal
Core has wrapped up (Alg/Cs (95%DD)) of 10 layers of wall material5Microcapsules.
(2) nano-ZnO, SiO2The load of particle
ZnO, SiO are taken respectively2Nanoparticle is dissolved in the water that pH is 4.0,7.0, the suspension of 10g/L is prepared into, in magnetic
30min is stirred on power blender.
Groped according to experiment condition, the loading condition for learning nano-ZnO is neutral environment and microcapsules outermost material is
Alg.Therefore, at room temperature, taking 50mL pH is 7.0 ZnO nanoparticle suspension, with (Cs(95%DD)/Alg)5Micro- glue
The microcapsules of loading ZnO nanoparticle can be obtained in capsule mixing, magnetic agitation 2h, and surface of microcapsule has loaded largely at this time
It nano-ZnO and is evenly distributed.SEM characterization is carried out to it, as shown in Figure 2 A.
Similarly, groped according to experiment condition, learn Nano-meter SiO_22Loading condition be acidic environment and microcapsules it is outermost
Layer material is Cs.Therefore, taking 50mLpH at room temperature is 4.0 SiO2Nanoparticle suspension, with microcapsules (Alg/
Cs (95%DD))5Load SiO can be obtained in mixing, magnetic agitation 2h2The microcapsules of nanoparticle, surface of microcapsule is negative at this time
A large amount of Nano-meter SiO_2 is carried2And it is evenly distributed.SEM characterization is carried out to it, as shown in Figure 2 B.
(3) nano-TiO2The load of particle
Due to nano-TiO2The dispersibility of particle is bad, and agglomeration easily occurs in adsorption process and cannot be adsorbed onto micro-
Capsule surface.So using nano-TiO2Experiment in, using from above two different method.With wall used in this experiment
Material Cs is as nano-TiO2The dispersing agent of particle.
Similarly, groped according to experiment condition, learn nano-TiO2Loading condition be and Cs formed compound improve point
The outermost material for dissipating property and microcapsules is Alg.Therefore take proper amount of nano TiO2- Cs (nano-TiO2With the compound of Cs) suspension
(pH value 5.0) and (Cs(95%DD)/Alg)5Microcapsules mixing, at room temperature, magnetic agitation 2h can be obtained load and receive
Rice TiO2The microcapsules of particle, final adsorption concentration are 300 mg/g.And contain nano-TiO to what is obtained2The microcapsules of particle into
Row SEM characterization, as shown in Figure 2 C.
Embodiment 2: Cry1Ac crystal microcapsules are investigated and resist ultraviolet ability
Studies have shown that crystalline protein stereochemical structure after ultraviolet irradiation is destroyed, to prevent crystalline protein from molten
In alkaline solution and insect midgut environment, therefore insecticidal effect can not be played.
This experiment closely and is equably wrapped in surface of microcapsule using nanoparticle as ultraviolet protective agent, to mention
The ability of high Cry1Ac crystalline protein ultraviolet radiation resisting.By the crystalline protein not wrapped up nanometer material different with having loaded three kinds
The microcapsules of material after natural air drying, are put into solar simulator in culture dish and irradiate the identical time.It is spent after taking out sample
Ion aqueous suspension leaves and takes a part and is used for biological activity determination, the insecticidal activity of insecticidal proteins remnants after detection ultraviolet irradiation
Just, the size of different nano material uvioresistant abilities is reflected with this.Another part carries out SDS-PAGE characterization, detects its purple
Alkali solubility after external exposure, and reflect with this size of uvioresistant ability of different nano materials.
Electrophoresis result is as shown in figure 3, the Cry1Ac crystal not wrapped up passes through 50mM Na after ultraviolet irradiation 2h2CO3(pH
10.2) solution carries out alkali solubility processing, and supernatant is taken to carry out SDS-PAGE (Fig. 3 A swimming lane 3), most of to be dissolved in high pH
Alkaline solution, residual protein amount be only without irradiation when 45%.And after irradiation time is more than 4h (Fig. 3 A swimming lane 4),
Cry1Ac crystalline protein cannot be dissolved in alkaline solution completely, this illustrates that the physicochemical property of the albumen has occurred that change,
Insect midgut environment cannot be re-dissolved in and then play insecticidal action.
Nano-meter SiO_2 is loaded2It is being irradiated after 8h with the Cry1Ac crystal microcapsules (Fig. 3 B/C) of ZnO, then by sample
Being dissolved in after being centrifuged in alkaline solution takes supernatant to carry out SDS-PAGE characterization, the results showed that (Fig. 3 D) is with ultraviolet irradiation time
Growth, although protein content has partial loss, but the alkali solubility of the albumen does not change, and loads nanometer through quantitative analysis
SiO2Retaining with the microcapsules albumen of ZnO is respectively 36% and 61%.And load nano-TiO2Microcapsules (Fig. 3 D) pass through 6h
After irradiation, then sample being dissolved in alkaline solution after centrifugation, supernatant is taken to carry out SDS-PAGE characterization, albumen reserved is 31%,
And albumen cannot be re-dissolved in alkaline solution substantially after ultraviolet irradiation 8h, show that the physicochemical property of albumen is destroyed.This demonstrate that this
Three kinds of nanoparticles all play the protective action of ultraviolet light irradiation to crystalline protein, and strongest ultraviolet protection effect is to receive
Rice ZnO, Nano-meter SiO_22Take second place, and nano-TiO2Effect is slightly poorer to the above two.
The measurement of embodiment 3:Cry1Ac bioactivity
(1) structure cell closes the LC of object and microcapsules50
Using Ostrinia furnacalis larvae as try insect, respectively to exposed crystal and the microcapsules for having loaded nanoparticle
Carry out insecticidal activity assay.The death toll for counting insect after culture 7d in larva optimum growh environment, utilizes SPSS software meter
Calculate its LC50, 95% confidence interval and regression equation etc., to determine whether being added for nanoparticle can be living to the desinsection of the dosage form
Property has an impact.
From the results shown in Table 1, parasporal crystal and its loaded nanoparticle microcapsules it is quick to Ostrinia furnacalis
There is preferable insecticidal activity in sense system.From numerically comparing for half lethal concentration, although the LC of microcapsules50Value is more mixed than structure cell
Close the LC of object50Want high.But its 95% confidence interval has overlay region, shows that microcapsules and structure cell mixture are all with higher
Insecticidal activity, and toxicity is without significant difference.
Meanwhile this experiment is also provided with negative control.As a result the lethality for being deionized water is 3.47%, nano-ZnO
The lethality of (50 ± 10nm) is 5.64%, nano-TiO2The lethality of (40 ± 10nm) is 6.25%, Nano-meter SiO_22(30±
Lethality 10nm) is 4.86%.The lethality of negative control group is lower, belongs within the scope of natural death, therefore can recognize
It customizes material used in standby process to act on the sensitive system's nonhazardous of Ostrinia furnacalis, this guarantees this experimental datas
Reliability and authenticity.
The LC of 1 Cry1Ac parasporal crystal of table and microcapsule formulations50
(2) influence of the microcapsules to the insecticidal activity of larva before and after ultraviolet irradiation
Microcapsule formulations without ultraviolet irradiation and irradiation exposed crystalline protein and load nano particle in different time periods
It is as shown in table 2 to the death rate of Ostrinia furnacalis larvae.
From table 2 it can be seen that exposed crystalline protein is being decreased obviously the lethality of larva after ultraviolet irradiation 2h
(P < 0.05), the irradiation of longer time influence the insecticidal activity of crystalline protein significant.
And the microcapsule formulations for having loaded nanoparticle can effectively weaken time of ultraviolet irradiation to larval mortality
It influences.The microcapsules for having loaded nano-ZnO still maintain higher insecticidal activity after ultraviolet irradiation 8h, with LC50When cause
Dead rate is without significant type difference (P > 0.05).Nano-meter SiO_2 is loaded2Microcapsule formulations lethality after ultraviolet irradiation 8h with
Untreated sample illustrates Nano-meter SiO_2 without significant type difference (P > 0.05)2The crystalline protein wrapped up in microcapsules is provided
Preferable ultraviolet protection effect, but it will be lower than microcapsule formulations of loading ZnO to the lethality of larva after irradiate 8 h.
Nano-TiO is loaded2Microcapsules within the time of ultraviolet irradiation 4h, to the lethality of larva and untreated sample without
Significant type difference (P > 0.05), but this dosage form is after time of ultraviolet irradiation is more than 6h, to the lethality of larva with do not locate
The sample of reason significantly decreases (P < 0.05).Illustrate nano-TiO2Killing for crystalline protein in microcapsules cannot be protected longer
Destruction of the worm activity from ultraviolet radioactive.From the above results, different nano materials can play purple to intracapsular crystalline protein
Outer protective action, but nano-ZnO and SiO2Protective effect be better than nano-TiO2。
Ultraviolet crystalline protein before and after the processing and Ostrinia furnacalis children of the microcapsules to survival for having loaded three kinds of nanoparticles
The weight influence of worm is listed in table 3.Compared with the exposed crystalline protein without ultraviolet irradiation, the crystal after 2h ultraviolet irradiation
Influence of the albumen to larval weight has had a significant type difference (P < 0.05), after ultraviolet irradiation, the larval weight of crystalline protein group
Increase and obviously illustrate that structure cell mixture weakens the inhibiting effect that larva grows, further demonstrates that the activity of crystalline protein is purple
External exposure destroys.Nano-ZnO and SiO are loaded2Microcapsules after ultraviolet irradiation 8h, the influence to larval weight is without significant
Type difference (P > 0.05), and loaded TiO2Influence significant (P of the microcapsules after ultraviolet irradiation 6h, to survival larval weight
<0.05).This illustrates that different nano materials can play the role of ultraviolet protection, but nano-ZnO and SiO to intracapsular crystalline protein2
Protective effect be better than nano-TiO2, this is consistent with the experimental result of larval mortality.
The above result shows that three kinds of nanoparticles provide effective ultraviolet protection to the crystalline protein in microcapsules
Effect.Wherein, the ultraviolet protection of nano-ZnO is all best on time and effect.Nano-meter SiO_22In ultraviolet photograph for a long time
It is slightly worse to the lethality of larva and growth inhibition effect after penetrating.And nano-TiO2It is most weak to the ultraviolet protection effect of crystalline protein.
This is consistent with the result of SDS-PAGE after ultraviolet processing.
The death rate of the ultraviolet crystalline protein before and after the processing of table 2 and microcapsule formulations to larva
The influence of the ultraviolet crystalline protein before and after the processing of table 3 and microcapsule formulations to survival larval weight
A. it is examined through independent sample T, sample does not have significant type difference (P > 0.05).
B. it is examined through independent sample T, sample has significant type difference (P < 0.05).
Embodiment 4:
Use method same as Example 1 that there is the Su Yunjin of uvioresistant ability using Cry1Ac crystal as core preparation
Bacillus microcapsules, unlike the first embodiment, the sequence of wall material from inside to outside are as follows: Cs-Alg-Cs-Alg-Cs-Alg-
Cs-Alg.Available (Cs (95%DD)/Alg) that 8 layers of wall material have been wrapped up using Cry1Ac crystal as core of the method4Micro- glue
Capsule.
Embodiment 5:
Use method same as Example 1 that there is the Su Yunjin of uvioresistant ability using Cry1Ac crystal as core preparation
Bacillus microcapsules, unlike the first embodiment, the sequence of wall material from inside to outside are as follows: Cs-Alg-Cs-Alg-Cs-Alg.
Available (Cs (95%DD)/Alg) that 6 layers of wall material have been wrapped up using Cry1Ac crystal as core of the method3Microcapsules.
Embodiment 6:
Use method same as Example 1 that there is the Su Yunjin of uvioresistant ability using Cry1Ac crystal as core preparation
Bacillus microcapsules, unlike the first embodiment, the sequence of wall material from inside to outside are as follows: Cs-Alg-Cs-Alg.The method can
Using (Cs (95%DD)/Alg) for obtaining having wrapped up 4 layers of wall material using Cry1Ac crystal as core2Microcapsules.
Using method identical with embodiment 2 and 3 to the Su Yunjin obtained by embodiment 4-6 with uvioresistant ability
Bacillus microcapsules carry out performance detection, the results showed that the microcapsules all have stronger insecticidal power, longer-term effect and compared with
Good resistant to high temperatures and resist drying stability, uvioresistant stability can reach 8 hours.Meanwhile the preparation cost of the microcapsules
It decreases.
It should be noted that embodiment described above for explaining only the invention, is not constituted to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair
It is bright to can be extended to other all methods and applications with the same function.
Claims (10)
1. a kind of thuringiensis microcapsules with uvioresistant ability, brilliant by the thuringiensis as core
Body protein matter, the cyst wall being coated on outside core, and it is adsorbed in the anti-ultraviolet nano material layer composition of cyst wall outer surface.
2. microcapsules according to claim 1, which is characterized in that the anti-ultraviolet nano material and thuringiensis
Mass ratio >=0.3:1 of microcapsules, preferably 0.3:1；And/or the anti-ultraviolet nano material includes nano-TiO2, nanometer
ZnO and Nano-meter SiO_22One or more of；And/or the thuringiensis microcapsules uvioresistant time >=2h, preferably
≥8h。
3. microcapsules according to claim 1 or 2, which is characterized in that the thuringiensis crystal proteins are
Cry1Ac crystal；And/or the cyst wall is big by the polyelectrolyte macromolecular with positive charge and the polyelectrolyte with negative electrical charge
Molecule is formed as the layer-by-layer alternating sorbent of wall material in the outer surface of core；Wherein, the polyelectrolyte with positive charge divides greatly
Attached bag includes chitosan and/or poly-D-lysine with positive charge, the preferably chitosan of positive charge；It is described with negative electrical charge
Macromolecular include one of negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate or
Several, preferably negatively charged sodium alginates.
4. microcapsules described in any one of -3 according to claim 1, which is characterized in that the wall material number of plies N of the cyst wall is
Integer more than or equal to 3, the wall material number of plies N of the preferably described cyst wall are the integer of 3-12, the wall of the further preferred cyst wall
Material number of plies N is the integer of 3-10；And/or the partial size of the microcapsules is (0.25-0.50 μm) × (0.60-1.00 μm)；Into one
Preferably, the anti-ultraviolet nano material is nano-ZnO and/or nano-TiO to step2, n-th layer wall material includes negatively charged seaweed
One or more of sour sodium, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate, preferably negatively charged seaweed
Sour sodium；Alternatively, the anti-ultraviolet nano material is Nano-meter SiO_22, n-th layer wall material includes with the chitosan of positive charge and/or more
Polylysine, the preferably chitosan of positive charge.
5. the preparation method of thuringiensis microcapsules described in any one of -4 according to claim 1 comprising negative
The step of carrying anti-ultraviolet nano material: the thuringiensis microcapsules that the wall material number of plies is N-1 or N are received with containing uvioresistant
The suspension mixing of rice material, stirring are obtained to load the thuringiensis microcapsules for having anti-ultraviolet nano material；It is preferred that
Ground, the content of anti-ultraviolet nano material is 10g/L in the suspension containing anti-ultraviolet nano material；And/or the anti-purple
Mass ratio >=0.3:1 of outer nano material and thuringiensis microcapsules, preferably 0.3:1.
6. according to the method described in claim 5, the anti-ultraviolet nano material is it is characterized in that, the wall material number of plies is N
Nano-ZnO and/or Nano-meter SiO_22, the suspension containing anti-ultraviolet nano material is dispersed in water by anti-ultraviolet nano material
It is formed.
7. according to the method described in claim 6, it is characterized in that, the anti-ultraviolet nano material is nano-ZnO, n-th layer wall
Material includes one or more of negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate and Sodium Hyaluronate,
Preferably negatively charged sodium alginate, the pH value of the suspension containing anti-ultraviolet nano material are 7.0, the stirring
Time is 2h；Alternatively, the anti-ultraviolet nano material is Nano-meter SiO_22, n-th layer wall material include with positive charge chitosan and/
Or poly-D-lysine, preferably the chitosan of positive charge, the pH value of the suspension containing anti-ultraviolet nano material are 4.0,
The time of the stirring is 2h.
8. according to the method described in claim 5, it is characterized in that, the wall material number of plies is N-1, the anti-ultraviolet nano material
For nano-TiO2, the suspension containing anti-ultraviolet nano material by anti-ultraviolet nano material and n-th layer wall material compound
It is dispersed in water to be formed；Wherein, the n-th layer wall material be include chitosan and/or poly-D-lysine with positive charge, preferably
For the chitosan of positive charge, N-1 layers of wall material include negatively charged sodium alginate, sodium carboxymethylcellulose, chondroitin sulfate
One or more of with Sodium Hyaluronate, preferably negatively charged sodium alginate；It is described containing anti-ultraviolet nano material
The pH value of suspension is 5.0, and the time of the stirring is 2h；Preferably, the compound of anti-ultraviolet nano material and n-th layer wall material
In, the mass ratio of the anti-ultraviolet nano material and n-th layer wall material is 1:1.
9. the method according to any one of claim 5-8, which is characterized in that the method also includes loading anti-purple
The step of thuringiensis microcapsules that the wall material number of plies is N-1 or N are prepared before the step of outer nano material comprising:
Step A, by the polyelectrolyte macromolecular with positive charge or negative electrical charge as wall material and the Su Yun as the first core material
Golden bacillus crystal proteins are uniformly mixed in salting liquid, concussion, in thuringiensis crystal proteins outer surface
First layer wall material is formed, after further separating treatment, washing, obtains the thuringiensis microcapsules that the wall material number of plies is 1；
Step B, by as wall material with wall material opposite charges in previous step polyelectrolyte macromolecular with as the
The thuringiensis microcapsules that the wall material number of plies of two core materials is 1 are uniformly mixed in salting liquid, and concussion is in the wall material number of plies
1 thuringiensis microcapsule exterior surface forms second layer wall material, after further separating treatment, washing, obtains wall material layer
The thuringiensis microcapsules that number is 2；
Step C is repeated in step B N-2 times, is often repeated once then one layer of wall material of increase, the Su Yun for being N thus to obtain the wall material number of plies
Golden bacillus microcapsules；
Wherein, the pH value of the salting liquid is 4.5-6.0, preferably 4.5-5.
10. any in the thuringiensis microcapsules or such as claim 5-9 as described in any one of claim 1-4
Application of the thuringiensis microcapsules of the preparation of method described in one in control of agricultural pest.
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| CN112385654A (en) * | 2020-09-16 | 2021-02-23 | 中国科学院武汉病毒研究所 | Anti-ultraviolet insect virus inclusion body particle and preparation method thereof |
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Granted publication date: 20210108 |